Streptococcal pyrogenic exotoxin B (SPE B) is a cysteine protease expressed by Streptococcus pyogenes. The D9N, G163S, G163S/A172S, and G239D mutant proteins were expressed to study the effect of the allelic variants on their protease activity. In contrast to other mutants, the G239D mutant was ϳ12-fold less active. The Gly-239 residue is located within the C-terminal S230-G239 region, which cannot be observed in the x-ray structure. The three-dimensional structure and backbone dynamics of the 28-kDa mature SPE B (mSPE B) were determined. Unlike the x-ray structure of the 40-kDa zymogen SPE B (proSPE B), we observed the interactions between the C-terminal loop and the active site residues in mSPE B. The structural differences between mSPE B and proSPE B were the conformation of the C-terminal loop and the orientation of the catalytic His-195 residue, suggesting that activation and inactivation of SPE B is involved in the His-195 side-chain rotation. Dynamics analysis of mSPE B and the mSPE B/inhibitor complexes showed that the catalytic and C-terminal loops were the most flexible regions with low order parameter values of 0.5 to 0.8 and exhibited the motion on the ps/ns timescale. These findings suggest that the flexible C-terminal loop of SPE B may play an important role in controlling the substrate binding, resulting in its broad substrate specificity. Streptococcus pyogenes (group A Streptococcus (GAS)3 ), one of the most common human bacterial pathogens, has developed diverse mechanisms that allow the bacteria to evade the immune system (1, 2). This bacteria causes a variety of human diseases, including pharyngitis, cellulitis, necrotizing fasciitis, streptococcal toxic shock syndrome, scarlet fever, acute rheumatic fever, rheumatic heart disease, and glomerulonephritis (3, 4). Virtually all strains of GAS isolated from patients with invasive disease express an extracellular cysteine protease known as streptopain (EC 3.4.22.10), with synonyms including streptococcal pyrogenic exotoxin B (SpeB or SPE B), streptococcus peptidase A, SPP, and streptococcal cysteine protease (SCP) (5-10). The level of SPE B activity is associated with the extent of gross pathological changes induced by the specific strains displaying varied degrees of virulence (5-7). Many reports also suggest that SPE B is an important virulence factor in streptococcal infections (11)(12)(13)(14). SPE B produced from GAS is released extracellularly to culture medium as a zymogen (proSPE B) with a molecular mass of 40 kDa. The conversion of proSPE B to the 28-kDa active mature SPE B (mSPE B) can be achieved by autoproteolysis and exogenous proteases (8 -10). SPE B has diverse substrate specificity and is involved in the processing of host proteins (10, 14). SPE B degrades extracellular matrix proteins fibronectin and vitronectin and increases bacterial attachment to host cells (6, 15). Additionally, it cleaves and activates matrix metalloproteases 2 and 9, which increase bacterial dissemination (16). It also causes severe inflammation in the host...

A technique of linear viscoelasticity measurements coupling with temperature scanning was found effective in the detection of microphase separation transition (MST) and in the determination of MST temperature. The validity and accuracy of the technique were confirmed and reinforced by atomic force microscopy and differential scanning calorimetry (DSC). The technique was applied to a study of the MST of a series of 13 polyurethane (PU) elastomers based on mixed toluene diisocyanate (TDI), 1,4 butadiol, and poly(tetramethylene oxide) (PTMO) of two different molecular weights; the MST temperatures of the PU elastomer samples were measured. Although each of the 13 polymer samples had distinct hard segment content and used PTMO of different chain lengths, or mixed PTMO, the MST temperatures of the 13 samples formed a linear master curve when the MST temperature was plotted against the fraction of hard segment. The master curve indicated that the MST temperature is independent of the length and type of PTMO. It was also found that 2,4 TDI prevailing over its isomer 2,6 TDI played a dominant role in the MST of this series of PU elastomers.

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